1. Field of the Invention
The present invention relates to improved methods for making small interfering RNA (siRNA), improved siRNA made by such methods, their use in the modulation of gene expression in mammalian and other cell types and their use in medical therapies.
2. Description of Related Art
RNA interference (RNAi) is a phenomenon in which a double stranded RNA (dsRNA) specifically suppresses the expression of a gene bearing its complementary sequence. The phenomenon was originally discovered in Caenorhabditis elegans by Fire and Mello (Fire et al., 1998). RNAi has since become a useful research tool for many organisms. Although the mechanism by which dsRNA suppresses gene expression is not entirely understood, experimental data provide important insights. In non-mammalian systems, it appears that longer dsRNA are processed into small, 21-23 nt dsRNAs by an enzyme containing RNase III motifs (Bernstein et al., 2001; Grishok et al., 2001; Hamilton and Baulcombe, 1999; Knight and Bass, 2001; Zamore et al., 2000). It has been theorized that the RNAi nuclease complex, called RNA-induced silencing complex (RISC), helps the small dsRNAs recognize complementary mRNAs through base-pairing interactions. Following the siRNAs interaction with its substrate, the mRNA is targeted for degradation, perhaps by enzymes that are present in the RISC (Montgomery et al., 1998).
Until recently, RNAi could only be used in non-mammalian cells. This is because mammalian cells have a potent antiviral response pathway that induces global changes in gene expression when the cells are challenged with long (>30 nucleotides) dsRNA molecules. This pathway has made it impossible to specifically suppress the expression of proteins in mammalian cells using the typical RNAi molecules, which are hundreds of nucleotides long.
Recently Elbashir et al. (2001) published a method to bypass the antiviral response and induce gene specific silencing in mammalian cells. Several 21 nucleotide (nt) dsRNAs with 2 nt 3′ overhangs were transfected into mammalian cells without inducing the antiviral response. These small dsRNAs, referred to as small interfering RNAs (siRNAs) proved capable of inducing the specific suppression of target genes. In one set of experiments, siRNAs complementary to a luciferase gene were co-transfected with a luciferase reporter plasmid into NIH3T3, COS-7, HeLaS3, and 293 cells. In all cases, the siRNAs were able to specifically reduce luciferase gene expression. In addition, the authors demonstrated that siRNAs could reduce the expression of several endogenous genes in human cells. The endogenous targets were lamin A/C, lamin B1, nuclear mitotic apparatus protein, and vimentin. The use of siRNAs to modulate gene expression in mammalian cells has now been repeated at least twice (Caplen et al., 2001; Hutvagner et al., 2001). This technology has great potential as a tool to study gene function in mammalian cells and may lead to the development of pharmacological agents based upon siRNA.
To realize this potential, siRNAs must be designed so that they are specific and effective in suppressing the expression of the genes of interest. Methods of selecting the target sequences, i.e. those sequences present in the gene or genes of interest to which the siRNAs will guide the degradative machinery, are directed to avoiding sequences that may interfere with the siRNA's guide function while including sequences that are specific to the gene or genes. Typically, siRNA target sequences of about 21 to 23 nucleotides in length are most effective. This length reflects the lengths of digestion products resulting from the processing of much longer RNAs as described above.
The making of siRNAs to date has been through direct chemical synthesis or through processing of longer, double stranded RNAs through exposure to Drosophila embryo lysates or through an in vitro system derived from S2 cells. Use of cell lysates or in vitro processing may further involve the subsequent isolation of the short, 21-23 nucleotide siRNAs from the lysate, etc., making the process somewhat cumbersome and expensive. Chemical synthesis proceeds by making two single stranded RNA-oligomers followed by the annealing of the two single stranded oligomers into a double stranded RNA. Methods of chemical synthesis are diverse. Non-limiting examples are provided in U.S. Pat. Nos. 5,889,136; 4,415,732; 4,458,066, expressly incorporated herein by reference, and in Wincott et al. (1995).
Elbashir and colleagues have published the procedure that they use to design, prepare, and transfect siRNAs for mammalian RNAi experiments. (“The siRNA user guide” Aug. 26, 2001). Similar protocols and procedures are available in Dharmacon Technical Bulletin #003, July 2001. These guides recommend chemically synthesizing two 21-mer RNA oligomers with two deoxythymidines at the 3′ terminus and 19 nucleotide complementary sequences. The two ribo-oligomers are mixed to allow them to hybridize. The products are then mixed with a transfection agent and added to cell culture at concentrations of about 100 nM. The pamphlet further recommends that the selection of the target sequence should be constrained so that they begin with AA and end with TT, so that the AA and TT overhang sequences may be fashioned from the target sequence itself. The pamphlet indicates that the symmetric 3′ overhangs aid the formation of approximately equimolar ratios of sense and antisense target RNA-cleaving siRNAs.
Several further modifications to siRNA sequences have been suggested in order to alter their stability or improve their effectiveness. It is suggested that synthetic complementary 21 mer RNAs having di-nucleotide overhangs (i.e. 19 complementary nucleotides+3′ non-complementary dimers) may provide the greatest level of suppression, although actual data demonstrating this advantage is lacking. These protocols primarily use a sequence of two (2′-deoxy) thymidine nucleotides as the di-nucleotide overhangs. These dinucleotide overhangs are often written as dTdT to distinguish them from the typical nucleotides incorporated into RNA. The literature has indicated that the use of dT overhangs is primarily motivated by the need to reduce the cost of the chemically synthesized RNAs. It is also suggested that the dTdT overhangs might be more stable than UU overhangs, though the data available shows only a slight (<20%) improvement of the dTdT overhang compared to an siRNA with a UU overhang.
To date, such chemically synthesized siRNAs are found to work optimally when they are in cell culture at concentrations of 25-100 nM. Elbashir et al. used concentrations of about 100 nM to achieve effective suppression of expression in mammalian cells. Unfortunately, ribo-oligomers are very expensive to chemically synthesize, making the procedure less appealing and not cost effective to many researchers and pharmaceutical companies. Furthermore, in foreseeable medical applications of siRNA, it would be desirable to achieve target gene inhibition with as little siRNA as possible. Therefore, siRNAs that are still as effective, if not more so, at lower concentrations would be significantly advantageous. There is therefore a need in the art for siRNAs that function at lower concentrations to modulate or attenuate the expression of target genes.
siRNAs have been most effective in mammalian cell culture at about 100 nM. In several instances, however, lower concentrations of chemically synthesized siRNA have been used. Caplen, et al. used chemically synthesized siRNAs at 18 nM. However, Caplen used semi-quantitative RT-PCR to monitor reduction of transcripts. The semi-quantitative nature of the assay makes unclear how great an effect this low concentration of siRNA had on transcript levels. Hutvagner, et al. used chemically synthesized siRNAs at concentrations of 70 nM to elicit a response. Although less than 100 nM, 70 nM may still represent a substantially prohibitive concentration for some applications. Although Elbashir et al. also indicated that they could use lower amounts of siRNA in the cell culture and still observe suppression, they did not provide data nor did they indicate by how much the expression was reduced at these lower levels.
WO 99/32619 and WO 01/68836 suggest that RNA for use in siRNA may be chemically or enzymatically synthesized. Both of these texts are incorporated herein in their entirety by reference. The enzymatic synthesis contemplated in these references is by a cellular RNA polymerase or a bacteriophage RNA polymerase (e.g. T3, T7, SP6) via the use and production of an expression construct as is known in the art. For example, see U.S. Pat. No. 5,795,715. The contemplated constructs provide templates that produce RNAs that contain nucleotide sequences identical to a portion of the target gene. The length of identical sequences provided by these references is at least 25 bases, and may be as many as 400 or more bases in length. An important aspect of this reference is that the authors contemplate digesting longer dsRNAs to 21-25mer lengths with the endogenous nuclease complex that converts long dsRNAs to siRNAs in vivo. They do not describe or present data for synthesizing and using in vitro transcribed 21-25 mer dsRNAs. No distinction is made between the expected properties of chemical or enzymatically synthesized dsRNA in its use in RNA interference.
Similarly, WO 00/44914, incorporated herein by reference, suggests that single strands of RNA can be produced enzymatically or by partial/total organic synthesis. Preferably, single stranded RNA is enzymatically synthesized from the PCR products of a DNA template, preferably a cloned cDNA template and the RNA product is a complete transcript of the cDNA, which may comprise hundreds of nucleotides. WO 01/36646, incorporated herein by reference, places no limitation upon the manner in which the siRNA is synthesized, providing that the RNA may be synthesized in vitro or in vivo, using manual and/or automated procedures. This reference also provides that in vitro synthesis may be chemical or enzymatic, for example using cloned RNA polymerase (e.g. T3, T7, SP6) for transcription of the endogenous DNA (or cDNA) template, or a mixture of both. Again, no distinction in the desirable properties for use in RNA interference is made between chemically or enzymatically synthesized siRNA.
U.S. Pat. No. 5,795,715 reports the simultaneous transcription of two complementary DNA sequence strands in a single reaction mixture, wherein the two transcripts are immediately hybridized. The templates used are preferably of between 40 and 100 base pairs, and which is equipped at each end with a promoter sequence. The templates are preferably attached to a solid surface. After transcription with RNA polymerase, the resulting dsRNA fragments may be used for detecting and/or assaying nucleic acid target sequences. U.S. Pat. No. 5,795,715 was filed Jun. 17, 1994, well before the phenomenon of RNA interference was described by Fire et al. (1998). The production of siRNA was, therefore, not contemplated by these authors.
As described above, there is a need for siRNAs of increased potency, both for general research and for use as medical or veterinary therapies. siRNAs of increased potency would decrease the risk or adverse reactions or other, undesired effects of medical therapies using siRNA. Fewer molecules of siRNA of increased potency would be needed for such therapies, with concomitant benefits to patients.